Enhanced Membrane Raft‐Redox Signaling Associated with NADPH Oxidase in Coronary Arterial Endothelium during Hypercholesterolemia

Background: The biological setting or location where reactive oxygen species (ROS) are produced is a new concept which may provide a mechanistic explanation for redox signaling specificity. In this regard, we recently reported that NADPH oxidase, a major superoxide producing enzyme system, is localized and functional in endothelial cell caveolae. Here, we investigate the role of caveolin-1, the main structural protein of caveolae, in the organization and regulation of NADPH oxidase activity and ROS mediated responses to cytokine challenge. Methods and Results: Superoxide production was measured by DHE assay in endothelial cells derived from wild type (wt) and caveolin-1 knockout (Cav−/ −) mouse lungs (MLEC). TNFα failed to induced superoxide production in Cav−/ − endothelial cell cultures (MOI = 0). However, infection of knockout cells with full-length caveolin-1 containing Adenovirus at MOI of 10 and 50 progressively restored TNFα effect on superoxide production with MOI 50 nearly reaching those observed in wt cells. The distribution of NADPH oxidase within the plasma membrane showed localization to membrane rafts fractions in wt cells which shifted to the non-raft membrane fractions in Cav-1−/ − MLEC. Re-expression of caveolin-1 (MOI 50) restored raft targeting of NADPH oxidase and both gp91 and p22phox co-precipitated with newly expressed caveolin-1 immunoprecipitated from raft fractions. To evaluate the cellular consequences of a potential redox signaling relay involving caveolin-1, we examined an NADPH oxidase dependent pathway involving activation of JNK1/2 MAP kinases and downstream upregulation of ICAM. We found that phosphorylation of JNK1/2 observed in wt MLEC in response to TNFα was significantly attenuated in Cav−/ − MLEC. In addition, TNFα did not induce upregulation of ICAM expression in caveolin-1 knockout MLEC or wt cells pretreated with DPI. Conclusions: Collectively, these results indicate that caveolin-1 is a necessary component of the molecular machinery for proper subcellular positioning required for cytokine activation of NADPH oxidase and superoxide production in endothelial cells. Thus, caveolin-1 may constitute a potential therapeutic target for altering ROS associated with cardiovascular pathology.

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Up-regulation of NADPH oxidase-mediated redox signaling contributes to the loss of barrier function in KRIT1 deficient endothelium

Scientific Reports ◽

10.1038/s41598-017-08373-4 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 27

Author(s):

Luca Goitre ◽

Peter V. DiStefano ◽

Andrea Moglia ◽

Nicholas Nobiletti ◽

Eva Baldini ◽

...

Keyword(s):

Nadph Oxidase ◽

Barrier Function ◽

Redox Signaling

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A novel pathway spatiotemporally activates Rac1 and redox signaling in response to fluid shear stress

The Journal of Cell Biology ◽

10.1083/jcb.201207115 ◽

2013 ◽

Vol 201 (6) ◽

pp. 863-873 ◽

Cited By ~ 44

Author(s):

Yunhao Liu ◽

Caitlin Collins ◽

William B. Kiosses ◽

Ann M. Murray ◽

Monika Joshi ◽

...

Keyword(s):

Nadph Oxidase ◽

Molecular Mechanisms ◽

Fluid Shear Stress ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Redox Signaling ◽

Hemodynamic Forces ◽

Rac1 Gtpase ◽

Embryonic Organ

Hemodynamic forces regulate embryonic organ development, hematopoiesis, vascular remodeling, and atherogenesis. The mechanosensory stimulus of blood flow initiates a complex network of intracellular pathways, including activation of Rac1 GTPase, establishment of endothelial cell (EC) polarity, and redox signaling. The activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can be modulated by the GTP/GDP state of Rac1; however, the molecular mechanisms of Rac1 activation by flow are poorly understood. Here, we identify a novel polarity complex that directs localized Rac1 activation required for downstream reactive oxygen species (ROS) production. Vav2 is required for Rac1 GTP loading, whereas, surprisingly, Tiam1 functions as an adaptor in a VE-cadherin–p67phox–Par3 polarity complex that directs localized activation of Rac1. Furthermore, loss of Tiam1 led to the disruption of redox signaling both in vitro and in vivo. Our results describe a novel molecular cascade that regulates redox signaling by the coordinated regulation of Rac1 and by linking components of the polarity complex to the NADPH oxidase.

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